Fig 2: SAM guide delivery approaches.a AAV83aTtr (n = 3) and AAV8GFP (n = 2) were delivered to R26SAM/+ mice. ELISA was employed to determine protein levels along with untreated R26SAM/+ (n = 2). Values were plotted as the mean per group +/- SD. Monthly p-values can be found in Supplementary Table 3. Two independent studies were conducted with similar results. b AAV84aTsx was delivered to R26SAM/+ mice (n = 3) to evaluate the tissue-specific induction of Tsx. Expression values relative to R26SAM/+ were plotted as the mean per group +/- SD. Liver: p = 0.001026; Spleen: p = 0.01138. c Protein induction by arrayed guide viral delivery to R26SAM/SAM mice was compared to single guide viral delivery (n = 3 for controls, n = 6 for treated). Serum levels were plotted as the mean per group +/- SD. p-values of each array comparison can be found in Supplementary Table 3. d AAV84aTsx comprises two guides targeting the 300 bp directly upstream of the TSS and another two guides targeting an upstream enhancer region (750 bp upstream). qPCR was completed on livers harvested from R26SAM/SAM treated with either AAV delivered guide array (4aTsx) or individual guides from the array (Tsx gA1-4) (n = 5). Expression values relative to R26SAM/+ were plotted as the mean per group +/- SD. p-values of each array comparison can be found in Supplemental Table 3. e LNP particles were formulated with synthetic Ttr gA3 SAM guides and introduced to R26SAM/SAM mice (n = 3). Protein expression levels were determined by ELISA with weekly bleeds. Daily p-values can be found in Supplementary Table 3. All values are plotted as mean +/- SD. Two independent replicates were conducted with similar results. f LNP particles were formulated with 0.5 mpk of synthetic Ttr gA2 SAM guide and introduced to R26SAM/SAM mice (n = 5) at zero weeks and/or 4 weeks. Protein expression levels were determined by ELISA with weekly bleeds. Daily p-values can be found in Supplementary Table 3. All values are plotted as mean +/- SD. g Overexpression by HDD delivery of Ttr cDNA or a plasmid expressing arrayed SAM guides was compared to AAV83aTtr in R26SAM/SAM mice (n = 5, per group). TTR levels were determined by ELISA and plotted as mean per group +/- SD. Untreated vs Array week 1: p = 0.000001; Untreated vs. Array week 2: p = 0.000001; Untreated vs HDD3aTtr week 1: p = 0.003585; Untreated vs. HDDTtr plasmid week 1: p = 0.002222. Two independent replicates were conducted with similar results. h A guide array expressing two activating guides each to Ngn3 and Btc was delivered to R26SAM/SAM mice (n = 2). Five tissues were collected and expression relative to R26SAM/SAM was plotted as mean per group +/- SD. Statistics: Asterisks (*) indicates significance, and the number of asterisks indicates the number of 0s after the decimal point. One-tailed, unpaired Student’s t-test for (a–j).

Fig 3: Tissue-specific overexpression in vivo.a LNPCre was delivered to R26LSL-SAM/+ to activate SAM expression in the liver (n = 3); data was plotted as the mean per group +/- SD. p-values of each comparison can be found in Supplementary Table 4. Three replicates were conducted with similar results. b A western blot showing dCas9SAM expression (219 kDa) in R26LSL-SAM/+ tissues treated with LNPCre. dCas9SAM signals are normalized to actin (41 kDa). One repeat was completed with similar results. c Expression profile of Tsx in R26LSL-SAM/4aTsx tissues with and without LNPCre treatment. Tsx expression relative to R26SAM/+ was determined by RT-qPCR (n = 3) and plotted as the mean per group +/- SD; p = 0.000082. d RNAscope of hypothalamus sections showing DAPI (blue), Pomc (green) and Ttr (magenta) in R26SAM/+ and WT mice (n = 2). e Zoom of ventromedial hypothalamus showing co-localization of Pomc and Ttr. Green arrows are pointing to white patches that represent co-localization. Additional individual repeats have not been conducted. f Graphical protocol for harvest and culture of the organ of Corti. g RT-qPCR determination of Ttr (p = 0.0015), Ldlr (p = 0.001395), and Pcsk9 (p = 0.000052) expression levels in organ of Corti explant cultures to R26SAM/SAM. Results are plotted as the mean per group +/- SD (n = 3). Asterisks (*) indicates significance, and the number of asterisks indicates the number of 0s after the decimal point. One-tailed, unpaired Student’s t-test for (a, c, g).

Fig 4: In vivo applications of R26SAM gene modulation.a Schematic of murine Ttr replaced with WT human TTR sequence. Mouse sequences are represented by blue lines and human sequences by purple. b Circulating mouse and human TTR levels were determined by species-specific ELISA and plotted as mean +/- SD (n = 8). Commercial normal human serum (NHS) was run as a control for normal human expression levels (n = 1). Humanized allele vs NHS: p = 0.0071. c Homozygous humanized TTR mice were crossed to R26SAM/SAM mice and injected with AAV83aTtr or AAV8GFP (n = 4). Asterisks (*) indicates significance, and the number of asterisks indicates the number of 0s after the decimal point. One-tailed, unpaired Student’s t-test for (b, c).